Exposure of the corneal epithelium to the environment leaves this important barrier susceptible to injury. Accordingly, the corneal epithelium has a remarkable self-renewal capacity. This self renewal is mediated by a reservoir of limbal stem cells which migrate to sites of injury where they proliferate and differentiate to replace the damaged epithelium. This proposal is directed toward understanding the role of 14-3-3C in corneal epithelial differentiation. 14-3-3 is a family of proteins that bind phosphoproteins and regulate their subcellular localization. 14-3-3C is the epithelial-specific family member, and using a mouse genetic model we demonstrate its crucial role in corneal epithelial differentiation Mutation of both copies of the gene leads to defective corneal epithelial development in the embryo. Heterozygous mutation allows for normal embryonic development;however, the corneal epithelial differentiation in response to injury remains impaired. Thus, 14-3-3C gene dosage distinguishes corneal epithelial development embryologically from corneal epithelial differentiation following injury. This defect in differentiation leads to outgrowth of undifferentiated cells which eventually form an opaque corneal plaque with associated inflammation and neovascularization. This plaque forms spontaneously as the heterozygous mice age, and we provide evidence that this is linked to a dry eye-like effect resulting from a defective meibomian gland which fails to secrete lipid. Notch1 is essential for corneal epithelial differentiation via its activation of genes important for both differentiation (including genes in the vitamin A pathway) as well as cell cycle arrest, and we have found that 14-3-3C mutant cells fail to activate Notch1. Expression of activated Notch1 restores differentiation in mutant cells, and accordingly the defective differentiation phenotype and the resulting corneal plaque formation closely resemble that seem with tissue-specific knockout of the Notch1 gene. We link 14-3-3C to cell surface expression of the EGF receptor (EGFR) and thus to the Erk signaling pathway which is activated by binding of EGF to this receptor. This is important because EGF signaling represses Notch1, and this signaling must be extinguished for Notch1 expression and subsequent corneal epithelial differentiation. We propose studies to further characterize the linkage between 14-3-3C, EGFR, and Notch1 (and its target genes) in corneal epithelia differentiation. These studies involve both cell culture and genetic cross using 14-3-3C mutant mice. PUBLIC HEALTH RELEVANCE: The integrity of the cornea, the most anterior part of the eye, is indispensable for vision. Forty-five million individuals worldwide are bilaterally blind and another 135 million have severely impaired vision in both eyes because of the loss of corneal transparency. Our proposed experiments aim to elucidate the essential role of 14-3-3C in corneal epithelial homeostasis and wound healing and to understand the molecular network that controls the process. We shall study the cornea epithelium development and wound healing process, barrier integrity, and the molecular/genetic properties of 14-3-3C mutant mice. We hope to better understand corneal disease development and provide new strategies for prevention and treatment.